The mechanisms underlying the exponentially increased incidence of breast cancer in women >55 years of age are poorly understood. A useful conceptual framework from which to build hypotheses is that ageing- related phenotypes are etiologically rooted in changes in tissue-specific stem cells or in their regulation. Indeed, common aging-related phenotypes i.e. cancers and deficits in tissue regeneration both have been linked to stem cells. A number of reports that studied stem cells as a function of age have demonstrated that age-related phenotypes are due to stem cell-intrinsic and -extrinsic factors, but that the delineation is tissue specific. The Bissell laboratory and others have shown that the mammary microenvironment is as important as are the mutations in epithelial tumor cells for development of breast cancers. In a number of cases it has even been shown that the microenvironment can be dominant over strong oncogenes. In the aging breast, does the microenvironment change so as to catalyze tumorigenesis? Do damaged or aged mammary stem cells cease listening to, or misinterpret regulatory cues from their microenvironment? Or is it a combination of the two? We are now uniquely poised to address these questions for the breast. Over several decades, 3-dimensional culture models that mimic many aspects of the human mammary gland and breast cancer microenvironments were developed in the Bissell laboratory. Recently, we also have developed a cell-based microenvironment microarray technology that facilitates elucidation of the functional roles that are played by individual microenvironmental constituents and combinations thereof. We have used these models together with primary human mammary progenitor cells to demonstrate that the microenvironment can dictate mammary stem cell fate decisions. Here we propose to combine these assets to address the following specific aims: (1) To identify age-dependent functional responses in microenvironment- directed mammary progenitor cell regulation, and the genetic circuitry that underlies them. (2) To determine whether mutations characteristic of breast cancers endow normal mammary progenitor cells with tumor-forming potential, or shifts their spectrum of response to mammary microenvironments in an age-dependent manner. (3) To design and test a therapeutic strategy based on age-related differences in mammary microenvironments and stem cell behavior using physiologically relevant 3D organotypic assays. RELEVANCE: As we age the risk of cancer rises and our body's tissues regenerate less efficiently;these characteristics of aging could be explained by dysregulated adult stem cells that otherwise maintain the affected tissues. Every cell resides in a so called 'microenvironment', which is known to direct cellular functions, and is known to change during the aging process. Here we are exploring how age-associated changes to the breast microenvironment and to gene expression could conspire to misdirect mammary stem cells to generate tumors.